| isolate | 'Prince' |
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| collection date | 2021-11-11 |
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| geographic location | USA: New Haven |
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| isolation source | phage display library |
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| sex | male |
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| age | not collected |
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| development stage | adult |
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| population | not applicable |
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| ethnicity | not applicable |
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| race | not applicable |
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| strain | not applicable |
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| disease | not applicable |
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| disease stage | not applicable |
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| treatment | not applicable |
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| tissue | peripheral blood lymphocytes |
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| health state | not applicable |
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| biomaterial provider | Barbara Kazmierczak, Yale University, Ste S140, 300 Cedar Street, New Haven, CT 06519 |
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| cell type | phage display |
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| synthetic | True |
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| age_event | sampling |
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| study_group_description | high-throughput cell-based phage display panning |
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| disease_length | not applicable |
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| prior_therapies | not applicable |
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| medical_history | not applicable |
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| anatomic_site | peripheral blood |
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| collection_time_point_reference | panning_start |
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| collection_time_point_relative | 8 |
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| collection_time_point_relative_unit | id: UO:0000033, label: day |
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| tissue_processing | phage elution by hydrochloric acid/triethylamine elution |
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| cell_phenotype | not applicable |
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| single_cell | False |
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| cell_number | not collected |
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| cells_per_reaction | not collected |
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| cell_storage | Y |
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| cell_quality | not collected |
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| cell_isolation | not applicable |
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| target_antigen | T3SS |
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| phage_display_bait_type | bacterial cells |
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| selection_name | 027i.1.E10.1 |
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| counter_selection_cell_genotype | ∆exsA |
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| counter_selection_cell_background | PA103 |
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| counter_selection_cell_strain_number | 219 |
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| counter_selection_cell_growth_condition | exponential phase |
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| counter_selection_cell_growth_protocol | a single colony was used to inoculate a culture of MinS media (25 mM potassium phosphate monobasic [KH~2~PO~4~], 95 mM NH~4~Cl, 110 mM disodium succinate, 50 mM monosodium glutamate [MSG], 1.25% v/v glycerol, 5 mM MgSO~4~, 18 µM FeSO~4~) which was grown shaking at 37 °C overnight, then subcultured 1/25 into the same media and grown for 3 hours. |
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| selection_cell_genotype | ∆exsD |
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| selection_cell_background | PA103 |
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| selection_cell_strain_number | 408 |
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| selection_cell_growth_condition | exponential phase |
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| selection_cell_growth_protocol | a single colony was used to inoculate a culture of MinS media (25 mM potassium phosphate monobasic [KH~2~PO~4~], 95 mM NH~4~Cl, 110 mM disodium succinate, 50 mM monosodium glutamate [MSG], 1.25% v/v glycerol, 5 mM MgSO~4~, 18 µM FeSO~4~) plus 10 mM nitrilotriacetic acid [NTA] which was grown shaking at 37 °C overnight, then subcultured 1/25 into the same media and grown for 3 hours. |
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| technical_replicate | 1 |
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| cell_processing_protocol | Library of phage-displayed VHHs were applied to counter-selection bacterial cells pre-washed with PBS+Ca+Mg and incubated 1 hour; bacteria removed by centrifugation 3750 rcf x 5 min, then supernatant applied to pre-washed selection bacterial cells and incubated 2 hours. Bacteria harvested by centrifugation, washed 4 times with PBS + Ca + Mg. Phage were eluted from bacterial cells by addition of 0.1 N HCl x 5 min, neutralization with 1 M Tris-HCl pH 11; bacteria harvested and second elution performed with 0.1 M triethylamine (TEA) x 5 min and neutralization with Tris pH 6.8. Acid- and base- eluates combined. One part eluate combined with nine parts exponential-phase E. coli culture and incubated 45 min at 37 °C; M13KO7 helper phage particles added to 1e10 pfu/mL final concentration and incubated 30 min; one part culture added to three parts media and incubated overnight. Cells removed by centrifugation 3750 rcf x 10 min; phage particles precipitated by addition of four volumes PBS + 0.5% BSA + 0.05% Tween-20 and one volume 20% w/v PEG-8000, 2.5 M NaCl to one volume supernatant and incubation on ice overnight; precipitated particles removed by centrifugation 4500 rcf x 1 hour, then resuspended in PBS + 0.5% BSA + 0.05% Tween-20. Phage concentration was ~1e13 pfu/mL. 2 µL of phage particles resuspended in PBS plus calcium and magnesium were used as template. |
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| immunogen | Four P. aeruginosa laboratory strains---PA14 (serotype O10), PAO1 (O5), PAK (O6) and PA103 (O11)---were grown to exponential phase in liquid LB and overnight on LB agar; cells were harvested, lysed by french press (>14,000 psi), and the lysate clarified by centrifugation. Membrane proteins were enriched by centrifugation through sucrose density gradients. Additionally, PA14 and PAO1 were grown as static biofilms in petri dishes in both LB and M9 minimal media at 30 ˚C for 36 hours, harvested by scraping, and lysed as above. The soluble proteins, enriched membrane proteins, and biofilm extracts were pooled to generate a mixed antigen preparation. |
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| phage_display_selection_round | 5 |
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| phage_display_selection_input_or_output | input |
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| phage_display_selection_selection_or_counterselection | selection |
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